Alternative splicing of precursor mRNAs (pre-mRNAs) is an important and widely conserved mechanism for increasing protein diversity and for gene regulation. In plants and other eukaryotes, tissue-specific and development-specific expression of genes is regulated by alternative splicing (1-3). Changes in pre-mRNA splicing of plant transcripts have been observed in response to stress conditions, including growth in cold temperature or under drought conditions (4). Alternative splicing also has been shown to contribute to diverse physiological processes in plants, including regulation of circadian rhythm and the defense response to pathogens (5, 6). Altogether, it has been estimated that between 20-30% of expressed genes are alternatively spliced in Arabidopsis thaliana and Oryza sativa (rice) (7, 8).
While alternative splicing appears to be extensively employed in natural regulatory systems, it has not been generally applied to the conditional expression of transgenes. Currently, transgene regulation is based almost exclusively on transcriptional activation. This is due, in no small part, to the ease of placing a promoter sequence upstream of any gene of interest, which requires little to no characterization of promoter elements and no alteration of the coding sequence. However, many conditional promoters suffer from issues such as leaky basal expression, pleiotropic effects, and species specificity (9). Promoters are also difficult to combine serially in order to generate complex regulatory patterns, as cross-talk between different promoter elements often leads to unpredictable effects on gene expression (10). Furthermore, use of multiple copies of identical promoters to coordinate regulation of several genes can trigger gene silencing (11). Thus, one reason for developing techniques based on alternative splicing for transgene regulation is that problems with existing conditional promoters may be ameliorated by combining DNA- and RNA-level regulation. Another advantage of a conditional splicing system is that the gene can be regulated and still remain under the control of its endogenous promoter.
Similar to conditional promoters that do not change the sequence of a translated ORF, alternative splicing of a cassette harboring a suicide exon can also be considered to operate in a traceless manner. Exon skipping would generate a productively translated spliced product (SP-I) with the suicide exon cleanly excised from the sequence of the ORF. Alternatively, exon inclusion would introduce a premature termination codon that targets the spliced product (SP-II) for nonsense-mediated decay (NMD) instead of undergoing translation (12). Thus, the presence of a suicide exon effectively eliminates gene expression, and its conditional splicing regulates expression of the encoded ORF. The coupling of alternative splicing to mRNA quality control pathways is conserved as a regulatory mechanism in diverse eukaryotic organisms, including plants, fungi, and metazoans (13), so this method for transgene regulation could have broad applicability.
Currently, only a few conditional splicing systems have been constructed that regulate gene expression in eukaryotic cells such as budding yeast (14) and mammalian cells (15, 16). These studies were performed either on single reporter constructs or in the context of gene fusions which introduced extraneous sequences to the N-terminus of the ORF, similar to minigene reporters used in splicing assays (17). In addition, a natural riboswitch has been discovered that regulates gene expression through alternative splicing in response to thiamine pyrophosphate (TPP) in plants and filamentous fungi (18-21). The untranslated regions (UTRs) containing the TPP riboswitch have been appended to reporter constructs. However, one problem with this riboswitch is that the level of gene activation is only modest even in thiamine-deficient plant lines, as levels of the spliced product which gives higher gene expression was increased ˜7-fold upon thiamine depletion (20). Thus, it has remained unclear whether conditionally spliced transgenes can be reliably designed for robust gene activation, such that this method is generalizable to any gene of interest.
Moreover, pre-mRNA splicing reactions can be sensitive to sequence context, as even single nucleotide polymorphisms have been shown to cause aberrant splicing in some genes (22). Thus, maintaining the fidelity and regulation of alternative splicing within diverse coding sequences can also be quite challenging.
Accordingly, a need exists for conditional splicing systems that provide robust gene activation for any gene of interest, and that maintain the fidelity and regulation of alternative splicing within diverse coding sequences.